Name: detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining
Uploaded: 2020-06-23T13:00:00.000+00:00
Duration: 4 min 53 s
Description: Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

This work was supported in part by the National Institutes of Health (K01DK114390), a Research Scholar Grant from the American Cancer Society (RSG-18-050-01-NEC), a Research Pilot Project Grant from University of New Mexico Environmental Health Signature Program and Superfund (P42 ES025589), a Shared Resources Pilot Project Award and a Research Program Support Pilot Project Award from UNM comprehensive cancer center (P30CA118100), and a new investigator award from the Dedicated Health Research Funds at the University of New Mexico School of Medicine.

1. Cell seeding
<ol>
	<li>Seed 2 x 105 HCT116 colorectal cancer cells per well in a 24-well plate and maintain the cells in Dulbecco&#39;s modified Eagle medium (DMEM) overnight at 37 &#176;C.</li>
	<li>Replace the culture medium with or without 100 &#181;M ferrous sulfate (FS) or 10 &#181;M doxorubicin (DOX) containing medium and incubate for 24 h.</li>
</ol>
2. Preparation of the DCFH-DA solution
<ol>
	<li>Dissolve 4.85 mg of DCFH-DA in 1 mL of dimethyl sulfoxide (DMSO) to make a 10 mM stock solution.</li>
	<li>Dilute the stock solution with pre-warmed DMEM into 10 &#181;M working solution right before adding it to the wells.</li>
	<li>Vortex the working solution for 10 s.</li>
</ol>
3. DCFH-DA staining
<ol>
	<li>Remove the drug containing medium and wash once with DMEM.</li>
	<li>Add 500 &#181;L of the DCFH-DA working solution into each well and incubate at 37 &#176;C for 30 min.</li>
	<li>Remove the DCFH-DA working solution. Wash once with DMEM and 2x with 1x phosphate-buffered saline (PBS).</li>
	<li>Add 500 &#181;L of 1x PBS to each well.</li>
</ol>
4. Imaging acquisition and intensity measurement
<ol>
	<li>Take representative fluorescent images for each well using the green fluorescent protein (GFP) channel on a fluorescence microscope.</li>
	<li>After taking images, remove PBS and add 200 &#181;L of radioimmunoprecipitation assay (RIPA) buffer to each well.</li>
	<li>Incubate on ice for 5 min, then collect cell lysate into 1.5 mL tubes.</li>
	<li>Centrifuge at 21,130 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer 100 &#181;L of the supernatant to a black 96 well plate and measure the fluorescence intensity using a fluorescence a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.</li>
	<li>Transfer 1 &#181;L of the supernatant to a clear 96 well plate containing 100 &#181;L of 1x protein assay solution to measure the protein concentration using the Bradford assay9.</li>
	<li>Normalize fluorescence intensities with protein concentrations.</li>
</ol>

<ol>
	<li>Birben, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+antioxidant+defense.">Oxidative stress and antioxidant defense.</a> World Allergy Organization Journal. 5 (1), 9-19 (2012).</li><li>Kim, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Oxidative+Stress+in+Neurodegenerative+Diseases.">The Role of Oxidative Stress in Neurodegenerative Diseases.</a> Experimental Neurobiology. 24 (4), 325-340 (2015).</li><li>Sullivan, L. B., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+and+cancer.">Mitochondrial reactive oxygen species and cancer.</a> Cancer &amp; Metabolism. 2, 17 (2014).</li><li>Formentini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ROS+Production+Protects+the+Intestine+from+Inflammation+through+Functional+M2+Macrophage+Polarization.">Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization.</a> Cell Reports. 19 (6), 1202-1213 (2017).</li><li>Rakotoarisoa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin-+and+Fish+Oil-Loaded+Spongosome+and+Cubosome+Nanoparticles+with+Neuroprotective+Potential+against+H2O2-Induced+Oxidative+Stress+in+Differentiated+Human+SH-SY5Y+Cells.">Curcumin- and Fish Oil-Loaded Spongosome and Cubosome Nanoparticles with Neuroprotective Potential against H2O2-Induced Oxidative Stress in Differentiated Human SH-SY5Y Cells.</a> ACS Omega. 4 (2), 3061-3073 (2019).</li><li>Mateen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Reactive+Oxygen+Species+Formation+and+Oxidative+Stress+in+Rheumatoid+Arthritis.">Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis.</a> PLoS One. 11 (4), (2016).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Hemin+and+Sestrin2+modulates+oxidative+stress+and+colon+tumor+growth.">The interaction of Hemin and Sestrin2 modulates oxidative stress and colon tumor growth.</a> Toxicology and Applied Pharmacology. 374, 77-85 (2019).</li><li>Wang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sotetsuflavone+inhibits+proliferation+and+induces+apoptosis+of+A549+cells+through+ROS-mediated+mitochondrial-dependent+pathway.">Sotetsuflavone inhibits proliferation and induces apoptosis of A549 cells through ROS-mediated mitochondrial-dependent pathway.</a> BMC Complementary and Alternative Medicine. 18, 235 (2018).</li><li>Kruger, N. J., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bradford+Method+For+Protein+Quantitation.">The Bradford Method For Protein Quantitation.</a> The Protein Protocols Handbook. , 17-24 (2009).</li><li>Tetz, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+the+dichlorofluorescein+assay+to+avoid+artifacts+in+measurement+of+toxicant-stimulated+cellular+production+of+reactive+oxidant+species.">Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species.</a> Journal of Pharmacological and Toxicological Methods. 67 (2), 56-60 (2013).</li><li>Rong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+detection+with+high+specificity+in+living+cells+and+inflamed+tissues.">Hydrogen peroxide detection with high specificity in living cells and inflamed tissues.</a> Regenerative Biomaterials. 3 (4), 217-222 (2016).</li><li>Liu, L. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+hydroxyl+radical+generated+in+quartz+powder/phosphate+buffer+solution+system+by+fluorescence+spectrophotometry.">Quantitative detection of hydroxyl radical generated in quartz powder/phosphate buffer solution system by fluorescence spectrophotometry.</a> Guang Pu Xue Yu Guang Pu Fen Xi. 34 (7), 1886-1889 (2014).</li></ol>

The authors have nothing to disclose.

The experimental protocol described here is easily reproducible to measure cellular total ROS. The critical steps include making DCFH-DA solution fresh and avoiding light exposure, minimizing cell status disturbance and extensive PBS washing right before taking images. For the preparation of DCFH-DA working solution, the stock solution should be added into pre-warmed DMEM right before adding into the 24 well plate. The reason is that old solutions that generate high background fluorescence or light exposure will lead to photobleaching. Most studies use 1x PBS or 1x Hanks&#39; balanced salt solution (HBSS) to dilute DCFH-DA and use it as reaction buffer10. However, when using HCT116 and RKO, dilution of DCFH-DA stock solution with PBS and fetal bovine serum free DMEM generated high background signal even in untreated cell. This may be due to cell status disturbance. In addition, the DCFH-DA working solution should be added slowly along the well wall. Disturbance of cell status will generate high fluorescence signal compared to the undisturbed nearby area. It is also critical to wash at least twice with PBS before taking images to reduce auto-fluorescence of the phenol containing DMEM. Phenol-free DMEM may be a better choice but we show here that PBS washing was sufficient to minimize auto-fluorescence. As shown in Figure 1, even in untreated control groups two different batches of experiments could result in different representative images. To control experimental variations, we recommend treating cells with diluted DCFH-DA working solution (as in the protocol) instead of adding stock solution directly onto the cells. Also, images should be taken in fields with similar cell densities and the same exposure time. Finally, it is important to perform experiments on all comparison groups at the same time.
Due to the significance of ROS, specific ROS detection, in addition to total ROS detection, has also been developed. For example, cellular production of superoxide can be detected by dihydroethidium, which upon oxidation results in hydroxylation at the 2-position to form 2-hydroxyethidium. As 2-hydroxyethidium intercalates into cellular DNA, red fluorescence with excitation and emission wavelengths of 535 nm and 635 nm, respectively, can be observed. Mitochondrial superoxide can be visualized with the MitoSOX reagent, a cationic derivative of dihydroethidium that enters live cells and specifically targets mitochondria. The oxidation product of Mitosox which generates red fluorescence can intercalates into mitochondrial DNA. Chemoselective fluorescent naphthylimide peroxide probe was developed for H2O2 detection11. In addition, detection of hydroxyl radicals using fluorescence spectrophotometry was also reported12.
In summary, here we described a simple and optimized protocol for detecting cellular total ROS using cost-effective DCFH-DA staining.

Three major reactive oxygen species (ROS) produced by cellular metabolism that are of physiological meaning are superoxide anion, hydroxyl radical, and hydrogen peroxide1. At low concentrations, they participate in physiological cell processes, but at high concentrations they have adverse effects on cell signaling pathways1. Our body has developed antioxidant systems, which are effective against excessive ROS. However, oxidative stress can occur when ROS overwhelm the detoxifying ability of our body, which contributes to many pathological conditions, including inflammation, cancer, and neurodegenerative disease2,3,4. The purpose of this method is to determine total cellular ROS in adherent cells using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The rationale is that oxidation of DCFH-DA to 2&#8217;-7&#8217;dichlorofluorescein (DCF) has been used extensively for total ROS detection including hydroxyl radicals (&#8226;OH) and nitrogen dioxide (&#8226;NO2). Mechanistically, DCFH-DA is taken up by cells where cellular esterase cleaves off the acetyl groups, resulting in DCFH. Oxidation of DCFH by ROS converts the molecule to DCF, which emits green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Compared with detection of fluorescence with flow cytometry and other alternative methods5, advantages of this method using a fluorescence microscope and a plate reader are that it produces clearly visible fluorescent images, and is easy to perform, efficient and cost-effective. This method has been widely used to detect cellular ROS for studying various conditions6,7,8. This protocol is used for detecting total ROS in adherent cells. Using this method to detect ROS in suspension cells may need some modifications.

<table><tbody><tr><td>2&#039;,7&#039;-Dichlorofluorescein diacetate</td><td>Cayman Chemical, Ann Arbor, MI</td><td>20656</td><td></td></tr><tr><td>Doxorubicin hydrochloride</td><td>TCI America, Portland, OR</td><td>D4193-25MG</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Corning, Corning, NY</td><td>45000-304</td><td></td></tr><tr><td>Ferrous Sulfate Heptahydrate</td><td>VWR, Radnor, PA</td><td>97061-542</td><td></td></tr><tr><td>Invitrogen EVOS FL Auto Imaging System</td><td>Thermo Fisher Scientific Waltham, MA</td><td>AMAFD1000</td><td>or any other fluorescence microscope</td></tr><tr><td>Protein assay Bradford solution</td><td>Bio-Rad, Hercules, CA</td><td>5000001</td><td></td></tr><tr><td>SpectraMax M2 Microplate Reader</td><td>Molecular Devices, Radnor, PA</td><td>89429-532</td><td>or any other fluorescence microplate reader</td></tr></tbody></table>

detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

HCT116 colorectal cancer cells were treated with 100 &#181;M FS or 10 &#181;M DOX to induce oxidative stress7. As shown in Figure 1, green fluorescence was dramatically increased by both FS and DOX as expected. To quantify the relative intensity change, the cells were lysed after taking images and normalized with protein concentrations. The quantified fluorescence intensity was significantly increased by FS or DOX in HCT116 cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60682/60682fig1.jpg" /> 
Figure 1: Iron treatment increases ROS in colorectal cancer cells. Representative fluorescent images taken by a fluorescence microscope and intensity quantification by a fluorescence microplate reader for 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in HCT116 cells after untreated control (Ctrl), (A) 100 &#181;M ferrous sulfate (FS) or (B) 10 &#181;M doxorubicin (DOX) treatment for 24 h. Scale bar = 400 &#181;m. * = p &#60; 0.05; *** = p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60682/60682fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

detection-total-reactive-oxygen-species-adherent-cells-2-7

Nanyang Technological University

Research

JoVE Journal

Biochemistry

30.0K Views.  University of New Mexico. Deze methode kan worden gebruikt om cellulaire reactieve zuurstofsoorten in aanhangende cellen met slechts een fluorescentiemicroscoop te analyseren en de intensiteit van de reactieve zuurstofsoorten te kwantificeren met fluorescentieplaatlezer. Dit protocol is eenvoudig, efficiënt en kosteneffectief. Begin met het zaaien van twee keer 10 tot de vijfde HCT116 colorectale kankercellen in DMEM in elke put van een 24-put plaat.Broed de cellen 's nachts bij 37 graden Celsius. Vervang de v...

Video: Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Medicine

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Immunology and Infection

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Biology

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

Bioengineering

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Neuroscience

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

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ROS Detection with DCFH-DA Staining: Measuring Total ROS in Colorectal Cancer Cell Lines

Source: Kim, H., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60682"> Detection of Total Reactive Oxygen Species in Adherent Cells by 2&#39;,7&#39;-Dichlorodihydrofluorescein Diacetate Staining.</a> J. Vis. Exp. (2020).
This video describes the protocol for detecting total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method helps visualize ROS localization in the adherent cells using a fluorescence microscope and further quantify ROS intensity with a fluorescence plate reader.

Source: Kim, H., et al.  Detection of Total Reactive Oxygen Species in Adherent Cells by 2&#39;,7&#39;-Dichlorodihydrofluorescein Diacetate Staining. J. ...

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

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